The Ras GTPase‐activating‐like protein IQGAP1 bridges Gasdermin D to the ESCRT system to promote IL‐1β release via exosomes

Abstract IL‐1β can exit the cytosol as an exosomal cargo following inflammasome activation in intestinal epithelial cells (IECs) in a Gasdermin D (GSDMD)‐dependent manner. The mechanistic connection linking inflammasome activation and the biogenesis of exosomes has so far remained largely elusive. Here, we report the Ras GTPase‐activating‐like protein IQGAP1 functions as an adaptor, bridging GSDMD to the endosomal sorting complexes required for transport (ESCRT) machinery to promote the biogenesis of pro‐IL‐1β‐containing exosomes in response to NLPR3 inflammasome activation. We identified IQGAP1 as a GSDMD‐interacting protein through a non‐biased proteomic analysis. Functional investigation indicated the IQGAP1‐GSDMD interaction is required for LPS and ATP‐induced exosome release. Further analysis revealed that IQGAP1 serves as an adaptor which bridges GSDMD and associated IL‐1β complex to Tsg101, a component of the ESCRT complex, and enables the packaging of GSDMD and IL‐1β into exosomes. Importantly, this process is dependent on an LPS‐induced increase in GTP‐bound CDC42, a small GTPase known to activate IQGAP1. Taken together, this study reveals IQGAP1 as a link between inflammasome activation and GSDMD‐dependent, ESCRT‐mediated exosomal release of IL‐1β.

I think the data presented are very interesting and propose a non-canonical role for GSDMD in IL-1 secretion (besides pore formation). The paper is well written, and the experiments are very well controlled. I think most of the conclusion presented are substantiated by the data presented (except detailed below).
My major concern is regarding the upstream events leading to IL-1 secretion in exosomes. We know that stimulation of cells with ATP leads to (despite other events) opening of the pannexin-1 channel by P2X7 stimulation and ion fluxes across the plasma membrane, which are responsible for inflammasome activation. The authors show the presence of cleaved IL-1 beta in the supernatant but cleavage of GSDMD can't be observed (or at least not shown in the study). We don't know if YAMC do activate NLRP3 and if all the processes shown are dependent on inflammasome activation, although the authors claim this in the text. Calcium influx across pannexin-1 channels can be envisioned to lead to activation of CDC42. IQGAP1 is, after all, a calcium responsive protein. Some experiment I would suggest to address questions concerning this knowledge gap in the study: 1. Use MCC950 or 90 mM extracellular KCl to check if secretion of exosomes is dependent on NLRP3 activation 2. Use zVAD-fmk or qVD to check if secretion is dependent on caspase-1 activation 3. Use extracellular EDTA or BAPTA-AM to check if exosome secretion or IQGAP1/GSDMD interaction is dependent on calcium influx Minor suggestions: Figure 3: Can the authors show a PLA assay between IQGAP1 ∆IQ and GSDMD as an independent confirmation of abolished interaction? Figure 4C/D: Quantification of PLA is missing. Is it possible to stain for pro-IL beta to see colocalization with CD63 in wildtype and IQGAP1 KO cells?
31st Mar 2022 1st Editorial Decision Dear Kate, Thank you for sending me your detailed point-by-point response of what experiments you can incorporate to address the raised concerns. I have now had a chance to take a careful look at everything and I appreciate the proposed experiments. Given this, I would therefore like to invite you to submit a revised version.
Let me know if we need to discuss anything further.
When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: https://www.embopress.org/page/journal/14602075/authorguide#transparentprocess We generally allow three months as standard revision time. As a matter of policy, competing manuscripts published during this period will not negatively impact on our assessment of the conceptual advance presented by your study. However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed. Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension.
Thank you for the opportunity to consider your work for publication. I look forward to your revision. Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide We realize that it is difficult to revise to a specific deadline. In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (29th Jun 2022). However, we can also easily extend this timeframe if needed. Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions. Use the link below to submit your revision: https://emboj.msubmit.net/cgi-bin/main.plex In this study Liao et al., report that IQGAP1 bridges GSDMD to the ESCRT system to promote IL-1β release via exosomes. The proposal is that IQGAP1 function as a bridge connecting GSDMD to ESCRT machinery to promote ubiquitinated-IL-1β and/or pro-IL-1β containing exosomes in response to inflammasome activation. The authors also propose that IQGAP1-GSDMD interaction recruits TSG101 on GSDMD+ late endosome to drive exosomal IL-1β release. In this context CDC42 serves as a molecular switch and enables IQGAP1 to bridge GSDMD to TSG101 during inflammasome-triggered exosome biogenesis.
Some of the proposed relatioships seem to be shown in isolation of other mainstream processes of biologically-active v release. The exosome relationships while tantalizing are quite teneous, and it is difficult to assess how significant they are. In the absence of showing that this pathway releases biologically active IL-1β engaged in signaling at target cells (the authors have no assays for activity of IL-1β) the significance of these observations is unclear. Major: 1. In the introduction, the authors sate that exosomes have been established as exit a mechanism of pyroptosis-associated release of IL-1β referencing a 2007 study. Much has happened since that time and gasdermin pores at the plasma membrane have been exceptionally well defined and accepted as the main pyroptotic mechanism of IL-1β release. The study, if it were to be published, needs to compare the exosomal cargo vs plasma membrane pore release to make sense 2. There are multiple problems with key data as outlined in points below, but the most important issue is the biological activity -if any -of the releasedIL-1β via this proposed exit pathway has not been addressed. The essays used are detecting IL-1β. But is it biologically active? This needs to be properly tested. Otherwise this is if anything a disposal pathway.
3. The interactions between IQGAP1 and GSDMD are shown by co-IPs nut no direct interaction has been established. Appropriate methods should be used to address and establish (rule in or out) that. Importantly, the IQGAP1 could be swept into large protein complexes with ESCRTs engaged in countering plasma membrane permeabilization, a well-established process.
4. Exosome and extracellular vesicles release accompany plasma membrane damage caused by GSDMD or other agents. This is a well known phenomenon, and yet the authors do not account or address any of that.
5. Others have shown that IQGAP1 in cooperation with TSG101 and other ESCRT protesin protects against GSDMD pores at the plasma membrane. It is possible that what the authors here are reporting what is actually a part of the previously published processes at the plasma membrane during GSDMD pore formation and pyroptotic plasmalemma permeabilization. This needs to be fully and formally addressed.
6. Further problems with key data: the 'endosomal localization' (?) in Fig. 4 is based on poor imaging and absence of quantification. (a) The only quantification is with EEA1 (early endosomal marker, not MVBs) in panel A, and not with CD63.
7. Along the same lines, the images in Fig4B with CD63 "show" some overlaps in yellow, but there are so many dots (background fluorescence ?) with IQGAP1 that one wonders if the authors were to flip by 180 degrees yellow and green images (randomizing) would the number of yellow dots remain the same... No quantification/statistics of any kind is a big issue here.
8. TSG101 is an upstream ESCRT in ESCRT machinery which recognizes the cargo protein but doesn't directly regulate the membrane remodeling. Did authors check other ESCRT proteins? 9. In the abstract the authors mention that IQGAP1 bridges GSDMD to ESCRT machinery to promote IL-1β containing exosomes in response to NLRP3 inflammasome activation but haven't shown any data on NLRP3 in this study.
10. Is IL-1β biologically active? 11. If increased in exosomes, how does IL-1β contact the receptors on cells to achieve its biliogical activity?
12. There are some known paradoxical relationship with GTP and GDP states known regarding IQGAPs and Rho GTPases. It seems that the authors may not be aware of those and believe that GTP state (typically associated with "active" small GTPases) is the ON state. This needs to be resolved. Also, IQGAPs are calmodcuin binding protins and Ca2+ may play a role...

Minor:
1. Please check the sizes of graphs in figure 3A 2. Figure 3E and F and figure 4F graphs are represented differently. 3. In figure 6A author haven't pointed out specific structure to look. 4. On any of the western blot author haven't shown the marker to represent the specific size of the proteins.

Referee #2:
Liao et al. (the authors) identify IQGAP1 as a potential interaction partner of gasdermin-D (GSDMD). They show that interaction between IQGAP1 and the C-terminal region of GSDMD is required for secretion of exosomes from YAMC cells upon treatment with LPS+ATP. These exosomes(or the intracellular complexes preceding secretion) contain IQGAP1, GSDMD and among other components TSG101 and activated CDC42. These complexes assemble in CD63 positive endosomes. Ablation of TSG101 expression or inhibition of CDC42 reduced secretion of exosomes upon LPS&ATP treatment. Furthermore, stable assembly of the CDC42, IQGAP1, GSDMD complex requires presence of both IQGAP1 or GSDMD, suggesting a stabilizing function for either proteins. The authors finish their study with the demonstration that exosome secretion is reduced in colonic explants from IQGAP1 or GSDMD KO treated with DSS.
I think the data presented are very interesting and propose a non-canonical role for GSDMD in IL-1 secretion (besides pore formation). The paper is well written, and the experiments are very well controlled. I think most of the conclusion presented are substantiated by the data presented (except detailed below).
My major concern is regarding the upstream events leading to IL-1 secretion in exosomes. We know that stimulation of cells with ATP leads to (despite other events) opening of the pannexin-1 channel by P2X7 stimulation and ion fluxes across the plasma membrane, which are responsible for inflammasome activation. The authors show the presence of cleaved IL-1 beta in the supernatant but cleavage of GSDMD can't be observed (or at least not shown in the study). We don't know if YAMC do activate NLRP3 and if all the processes shown are dependent on inflammasome activation, although the authors claim this in the text. Calcium influx across pannexin-1 channels can be envisioned to lead to activation of CDC42. IQGAP1 is, after all, a calcium responsive protein. Some experiment I would suggest to address questions concerning this knowledge gap in the study: 1. Use MCC950 or 90 mM extracellular KCl to check if secretion of exosomes is dependent on NLRP3 activation 2. Use zVAD-fmk or qVD to check if secretion is dependent on caspase-1 activation 3. Use extracellular EDTA or BAPTA-AM to check if exosome secretion or IQGAP1/GSDMD interaction is dependent on calcium influx Minor suggestions: Figure 3: Can the authors show a PLA assay between IQGAP1 ∆IQ and GSDMD as an independent confirmation of abolished interaction? Figure 4C/D: Quantification of PLA is missing. Is it possible to stain for pro-IL beta to see colocalization with CD63 in wildtype and IQGAP1 KO cells?
PART I: Key Conceptual and Technical Issues Raised by the Reviewers.
Conceptual Issues: 1. The biological activity of the IL-1 released through the exosomal pathway.
Compared to mature IL-1 (~17kDa), pro-IL-1 (~31kDa) retains a propeptide and therefore has a much lower affinity and potency for the IL-1 receptor (IL-1R). Pro-IL-1 is converted to mature IL-1 upon inflammasome activation via proteolytic cleavage by caspase 1 or caspase 8. Since our data showed that the it was the pro-IL-1 that was recognized by the ESCRT system as an exosomal cargo protein, it is natural astute for the Reviewer 1 to question whether the IL-1 released through the exosomal pathway is biologically active (major points #2 and #10).
We actually found that active caspase 8 as well as other inflammasome components were co-released with polyubiquitinated pro-IL-1. These data were not included in the original submission for simplicity. Nevertheless, it is important to note that the exosomal release of IL-1 is driven by activated inflammasome capable of processing pro-IL-1. Consistent with the presence of active caspase 8, we did detect mature IL-1 in LPS and ATP-induced exosomes from YAMC cells along with polyubiquitinated pro-IL-1 (Fig.1A, 3D, 4F, as indicated by prominent bands around 17kDa). Collectively, the evidence strongly supports the notion that exosomal IL-1 is biologically active.
To further validate the bioactivity of exosomal IL-1, we tested whether exosome preparations can activate the hallmark pathway of IL-1 signaling, NFB, in a luciferase reporter cell line (  Exosomal proteins concentrated from 10 7 exosomes were prepared from supernatants of YAMCs that were left untreated or treated with LPS (4hrs) and ATP (30min). HEK293 cells stably expressing IL-1R1 were transfected with an NFκB luciferase reporter construct and stimulated with indicated exosomal proteins in the presence or absence of anti-IL-1β overnight. Cell lysates were analyzed for luciferase activity. B. Exosome preparations from colon explants of DSS-treated wild-type (WT) and Il1b-/-mice were subjected to IL-1β bioactivity assay. Exosomal proteins concentrated from 10 7 exosomes were prepared from supernatants of the colon explants. HEK293 cells stably expressing IL-1R1 were transfected with an NFκB luciferase reporter construct and stimulated with indicated exosome preparations in the presence or absence of anti-IL-1β overnight. Cell lysates were analyzed for luciferase activity. ***, P<0.001 by unpaired two-tailed t test. All experiments were repeated 3 times with consistent results, the representative results are shown.
We enriched for exosomal proteins using Amicon Ultra Centrifugal Filters and utilized a commercial IL-1β activity reporter cell line to measure IL-1β biological activity (Magupalli et al., 2020). An NFκB luciferase reporter (Schindler & Baichwal, 1994) was transfected into a HEK293 cell line that stably overexpresses IL-1R1. Six hours after transfection, concentrated exosome preparations were added to transfected cells. Stimulated cells were lysed and cell lysates were subjected to luciferase activity assay. Exosome preparations from LPS and ATP-treated YAMCs induced about 4-fold increase in luciferase activity, which was abolished in the presence of anti-IL-1β neutralizing antibody (Fig. IA). Similarly, exosome preparations from DSS-challenged wild-type but not Il1b knockout mouse colon explants also induced over 5-fold increase in luciferase activity (Fig. IB).
As Reviewer 1 referenced in major points #1 and #4, GSDMD-constituted pores in, and the subsequent rupture of plasma membrane was perceived as a major route of exit for IL-1 when GSDMD was first identified as a pyroptosis executor. However, our understanding has rapidly evolved over the last few years, with increasing evidence demonstrating that IL-1 can be released independent of plasma membrane rupture especially in nonmyeloid cells such as neutrophils, dendritic cells as well as so-called hyperactivated macrophages (Evavold, Ruan et al., 2018) (Karmakar, Minns et al., 2020) (Zanoni, Tan et al., 2016).
To more conclusively determine whether formation of GSDMD pores contributes to exosomal release of IL-1in YAMCs, we tested whether GSDMD I105N, a mutant that cannot constitute membrane pores (Kayagaki, Stowe et al., 2015), is capable of mediating the biogenesis of IL-1-containing exosomes. The GSDMD I105N mutant is a perfect control for the purpose in that we have shown that this mutation does not affect GSDMD-IQGAP1 interaction (Fig. 2F). GSDMD I105N showed comparable capacity in mediating the release of poly-ubiquitinated pro-IL-1β ( Fig. IIA in this document, Fig. EV1D in the manuscript), indicating that the pyroptotic activity of GSDMD is dispensable for exosomal IL-1β release. Consistently, the production of polyubiquitinated pro-IL-1did not associate with an increased in propidium iodide (PI) (Fig. IIB, and Fig. EV1E in the manuscript), a finding in line with our previous report (Bulek, Zhao et al., 2020).
Western blot analysis of supernatants and whole cell lysates collected from GSDMD KO YAMC cells expressing either wild-type of I105N GSDMD stimulated with or without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes). B. GSDMD KO YAMC cells expressing either wild-type of I105N GSDMD were stimulated with or without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) n phenol red-free DMEM supplemented with 1 µg/ml PI.

The dual functions of ESCRT in mediating membrane repair and in exosome biogenesis.
As another point related to membrane rupture, Reviewer 1 referenced the recent discovery that ESCRTmediated membrane repair, a process also assisted by IQGAP1, is triggered by GSDMD pores. The Reviewer thus questioned whether our observation is merely another rendition of the same cellular process (major point #5). We are aware of the referenced studies and have discussed in detail the similarity and distinction of these two pathways in the manuscript. We respectfully disagree with this alternative interpretation suggested by the Reviewer. If the ESCRT system had been activated to promote membrane repair in response to LPS and ATP stimulation in the YAMC cells, blockade of its activation, by either knocking out IQGAP1 or knocking down Tsg101 as we did in the study, should have led to enhanced cell death and thereby more IL-1 release. On the contrary, we instead found that inhibition of ESCRT prevented exosomal IL-1 release, thus conclusively ruling out the activation of ESCRT-mediated membrane repair in our experimental system.
That said, our findings, in light of the prior reports, do beg the question of what determines the role of ESCRT (membrane repair vs. exosome formation). Of note, studies that analyzed the role of ESCRT in membrane repair (Claude-Taupin, Jia et al., 2021, Ruhl, Shkarina et al., 2018 invariably employed stimulations that directly damage the plasma membrane, including treatment with a gentle detergent (Claude-Taupin et al., 2021), overexpressing the N-terminal fragment of GSDMD (Ruhl et al., 2018), or transfection of LPS (Claude-Taupin et al., 2021, Ruhl et al., 2018 which directly activates caspase 11 independent of NLRP3 inflammasome. Consistent with this prior report, unstimulated IQGAP1-deficient YAMCs appeared more sensitive to plasma membrane damage induced by low-dose saponin, as shown by increased PI uptake and LDH release compared to wild-type cells ( Fig. EV3D). This result underscores the fact that the exosomal pro-IL-1β release, driven by NLRP3-inflammasome activation, is an active process involving cargo protein selection, rather than a passive discharge of cytoplasmic contents. Furthermore, the data suggest that LPS and ATP stimulation is required to engage the ESCRT system for exosome biogenesis. The differences in cell types and stimuli, which dictate how and where the ESCRT system is engaged, likely account for the divergent observations.

The involvement of inflammasome in IQGAP1-GSDMD-mediated exosomal IL-1 release.
Both Reviewer 1 (major point #9) and Reviewer 2 (major points #1 and #2) astutely noted that additional evidence is needed to substantiate our claim that exosomal IL-1 release is driven by the NLRP3 inflammasome. We agree with the reviewers wholeheartedly on this matter.
MCC950, a small molecule that inhibits NLRP3 oligomerization, we found that inhibition of NLRP3 inflammasome activation abolished the production of poly-ubiquitinated pro-IL-1 and sEVs production in response to LPS and ATP stimulation (  5. The involvement of calcium signaling in IQGAP1-GSDMD-mediated exosomal IL-1 release. Both Reviewer 1 (major point #12) and Reviewer 2 (major point #3) insightfully pointed out the crucial role of calcium signaling in the regulation of IQGAP1. Indeed, calcium flux is induced by ATP stimulation and critically contributes to NLRP3 inflammasome activation. We agree with both reviewers and would like to formally test the involvement of calcium signaling in IQGAP1-GSDMD-mediated exosomal IL-1 release.
Following the reviewer's suggestion, we evaluated the impact of calcium chelation on exosomal release of pro-IL-1β. We found that BAPTA-AM treatment decreased the production of polyubiquitinated pro-IL-1β ( Fig. V in  this document, Fig. EV3E). Intracellular calcium mobilization has been long recognized as a trigger for ESCRT activation (Scheffer, Sreetama et al., 2014), which signals through calcium-binding protein-apoptosis-linked gene (ALG). Thus, the data from BAPTA-AM treatment serves to corroborate the involvement of ESCRT system in LPS and ATP-induced exosomal IL-1β release. Figure V. Western blot analysis of supernatants and whole cell lysates collected from YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) in the presence or absence of BAPTA-AM (6µM).

A direct interaction between IQGAP1 and GSDMD.
We identified the IQGAP1-GSDMD interaction using co-immunoprecipitation, which detects not only direct physical interaction but also complexes that are formed via mutual binding to a third component or even lipids. Reviewer 1 requested that we rule out or rule in a direct interaction between IQGAP1 and GSDMD to help discern at which stage IQGAP1 may be involved (major point # 3). In addition to co-immunoprecipitation, we showed additional evidence for the IQGAP1-GSDMD interaction using proximity ligation assay (Fig. EV2) We agree with the reviewer that the presented interaction using co-immunoprecipitation data alone does not prove a direct interaction between IQGAP1 and GSDMD. We realized that this is one of the limitations of our study and will include this discussion in the text. Since IQGAP1 is 190kD, it will be technically challenging to test the direct interaction with the recombinant proteins. Since both proteins are required for the same function (IL1β release), our results collectively support their functional interaction with either direct or indirect physical interaction in a complex.
We showed that Tsg101, which not only recognizes cargo substrate but also recruits downstream components of the ESCRT system, was required for IQGAP1-GSDMD-mediated exosomal IL-1 release. Reviewer contended that Tsg101 is an upstream factor that does not directly participate in membrane remodeling (major point # 8). To our knowledge there is very little information on any ESCRT-independent function of Tsg101, thus our data suggest that it is the ESCRT system that mediates the IQGAP1-GSDMD-mediated exosomal IL-1 release. However, we agree with the reviewer that additional evidence would further support its involvement in the process.
Knockdown of Vps24, a component of ESCRT-3 that catalyzes the last step of membrane remodeling during ILV formation, suppressed the release of polyubiquitinated pro-IL-1β and extracellular vesicles (Fig. VI in this  document, Fig. EV3A-B), further demonstrating a critical role of the ESCRT system in this process. with either scramble or Vsp24-targeting siRNA followed by stimulation with LPS for 4 hours 48 hours post transfection. After LPS stimulation, cells were further exposed to ATP for 30 minutes. Supernatants from cells treated described in panel A were subjected to methanol treatment to precipitate total protein, which were then solubilized with Laemmli buffer and analyzed by Western blot. B. Nanoparticle tracking analysis of supernatant of scramble siRNA or Vsp24 targeting siRNA transfected YAMC cells treated with LPS plus ATP. *, P<0.05 by unpaired two-tailed t test.
Technical Issues: 1. Image quality and quantification. Reviewer 1 pointed out that the evidence we provided for endosomal localization of IQGAP1 and GSDMD is unsatisfactory, primarily because of inadequate quantification (major points #6 and #7). We thank the reviewer for noting the deficiencies. We have re-analyzed our confocal images and specifically quantified the extent of signal co-localization with CD63. The reviewer also noted that the IQGAP1 signal appears to be excessively abundant and questioned whether the signal was autofluorescence. We have in fact had a control staining using IQGAP1 knockout cells and verified that the detected signals were indeed specific to IQGAP1 ( 2. Additional PLA and immunofluorescent staining to validate interaction and localization of IL-1. Reviewer 2 requested additional proximal ligation assay (PLA) between IQGAP1 ∆IQ and GSDMD as an independent confirmation of abolished interaction. In addition, the reviewer also requested to visualize pro-IL-1 in colocalization with CD63 in wild-type and IQGAP1 KO cells. We performed the prescribed PLA and include the data in revision (Figs. 3C). The PLA confirmed the loss of interaction between IQGAP1 ∆IQ and GSDMD. We also performed the suggested staining and found that IQGAP1 deficiency alters pro-IL-1 trafficking to CD63+ puncta (Fig. 4E).
PART II: Point-by-point response

Referee #1
In this study Liao et al., report that IQGAP1 bridges GSDMD to the ESCRT system to promote IL-1β release via exosomes. The proposal is that IQGAP1 function as a bridge connecting GSDMD to ESCRT machinery to promote ubiquitinated-IL-1β and/or pro-IL-1β containing exosomes in response to inflammasome activation. The authors also propose that IQGAP1-GSDMD interaction recruits TSG101 on GSDMD+ late endosome to drive exosomal IL-1β release. In this context CDC42 serves as a molecular switch and enables IQGAP1 to bridge GSDMD to TSG101 during inflammasome-triggered exosome biogenesis.
Some of the proposed relationships seem to be shown in isolation of other mainstream processes of biologically-active v release. The exosome relationships while tantalizing are quite teneous, and it is difficult to assess how significant they are. In the absence of showing that this pathway releases biologically active IL-1β engaged in signaling at target cells (the authors have no assays for activity of IL-1β) the significance of these observations is unclear. Major: 1. In the introduction, the authors sate that exosomes have been established as exit a mechanism of pyroptosis-associated release of IL-1β referencing a 2007 study. Much has happened since that time and gasdermin pores at the plasma membrane have been exceptionally well defined and accepted as the main pyroptotic mechanism of IL-1β release. The study, if it were to be published, needs to compare the exosomal cargo vs plasma membrane pore release to make sense.
GSDMD-constituted pores in, and the subsequent rupture of plasma membrane was perceived as a major route of exit for IL-1 when GSDMD was first identified as a pyroptosis executor. However, our understanding has rapidly evolved over the last few years, with increasing evidence demonstrating that IL-1 can be released independent of plasma membrane rupture especially in non-myeloid cells such as neutrophils, dendritic cells as well as so-called hyperactivated macrophages (Evavold, Ruan et al., 2018) (Karmakar, Minns et al., 2020) (Zanoni, Tan et al., 2016).
To more conclusively determine whether formation of GSDMD pores contributes to exosomal release of IL-1in YAMCs, we tested whether GSDMD I105N, a mutant that cannot constitute membrane pores (Kayagaki, Stowe et al., 2015), is capable of mediating the biogenesis of IL-1-containing exosomes. The GSDMD I105N mutant is a perfect control for the purpose in that we have shown that this mutation does not affect GSDMD-IQGAP1 interaction (Fig. 2F). GSDMD I105N showed comparable capacity in mediating the release of poly-ubiquitinated pro-IL-1β (Fig. EV1D), indicating that the pyroptotic activity of GSDMD is dispensable for exosomal IL-1β release. Consistently, the production of polyubiquitinated pro-IL-1did not associate with an increased in propidium iodide (PI) (Fig. EV1E), a finding in line with our previous report (Bulek, Zhao et al., 2020).
2. There are multiple problems with key data as outlined in points below, but the most important issue is the biological activity -if any -of the releasedIL-1β via this proposed exit pathway has not been addressed. The essays used are detecting IL-1β. But is it biologically active? This needs to be properly tested. Otherwise this is if anything a disposal pathway.
To further validate the bioactivity of exosomal IL-1, we tested its ability to activate the hallmark pathway of IL-1 signaling, NFB, in a luciferase reporter cell line. We prepared exosomal proteins from LPS and ATPstimulated YAMC cells and colon explants to stimulate an NFB reporter cell line. This assay provided a specific and robust readout for IL-1 activity, which demonstrated that exosomal proteins contain biologically active IL-1β ( Fig. EV1A and F).
3. The interactions between IQGAP1 and GSDMD are shown by co-IPs nut no direct interaction has been established. Appropriate methods should be used to address and establish (rule in or out) that. Importantly, the IQGAP1 could be swept into large protein complexes with ESCRTs engaged in countering plasma membrane permeabilization, a well-established process.
We identified the IQGAP1-GSDMD interaction using co-immunoprecipitation, which detects not only direct physical interaction but also complexes that are formed via mutual binding to a third component or even lipids. Reviewer 1 requested that we rule out or rule in a direct interaction between IQGAP1 and GSDMD to help discern at which stage IQGAP1 may be involved. In addition to co-immunoprecipitation, we showed additional evidence for the IQGAP1-GSDMD interaction using proximity ligation assay (Fig. EV2A-C) We agree with the reviewer that the presented interaction using co-immunoprecipitation data alone does not prove a direct interaction between IQGAP1 and GSDMD. We realized that this is one of the limitations of our study and will include this discussion in the text. Since IQGAP1 is 190kD, it will be technically challenging to test the direct interaction with the recombinant proteins. Since both proteins are required for the same function (IL-1-β release), our results collectively support their functional interaction with either direct or indirect physical interaction in a complex.
Regarding whether IQGAP1 plays a role in countering membrane repair during NLRP3 inflammasome activation in our system, please see our responses to point #4 and 5.
4. Exosome and extracellular vesicles release accompany plasma membrane damage caused by GSDMD or other agents. This is a well-known phenomenon, and yet the authors do not account or address any of that. 5. Others have shown that IQGAP1 in cooperation with TSG101 and other ESCRT protein protects against GSDMD pores at the plasma membrane. It is possible that what the authors here are reporting what is actually a part of the previously published processes at the plasma membrane during GSDMD pore formation and pyroptotic plasma membrane permeabilization. This needs to be fully and formally addressed.
Since the questions #4 and 5 are related, we will address them together in this section.
We are aware of the referenced studies and have discussed in detail about the similarity and distinction of these pathways in the manuscript. For the reasons listed below, we respectfully disagree with the Reviewer about the membrane repairing mechanism as an alternative interpretation for our study. If the ESCRT system had been activated to promote membrane repair in response to LPS and ATP stimulation in the YAMC cells, blockade of its activation, by either knocking out IQGAP1 or knocking down Tsg101 as we did in the study, should have led to enhanced cell death and thereby more IL-1β release. On the contrary, we instead found that inhibition of ESCRT prevented exosomal IL-1β release, thus conclusively ruling out the activation of ESCRT-mediated membrane repair in our experimental system. Consistent with this prior report, unstimulated IQGAP1-deficient YAMCs appeared more sensitive to plasma membrane damage induced by low-dose saponin, as shown by increased PI uptake and LDH release compared to wild-type cells (Fig. EV3C). Notably, the membrane damage induced by low dose saponin did not lead to the release of polyubiquitinated pro-IL-1β in unstimulated YAMCs (Fig. EV3D). This result underscores the fact that the exosomal pro-IL-1β release, driven by NLRP3-inflammasome activation, is an active process involving cargo protein selection, rather than a passive discharge of cytoplasmic contents. Furthermore, the data suggest that LPS and ATP stimulation is required to engage the ESCRT system for exosome biogenesis. The differences in cell types and stimuli, which dictate how and where the ESCRT system is engaged, likely account for the divergent observations.
6. Further problems with key data: the 'endosomal localization' (?) in Fig. 4 is based on poor imaging and absence of quantification. (a) The only quantification is with EEA1 (early endosomal marker, not MVBs) in panel A, and not with CD63.
We thank the reviewer for noting the deficiency. We re-analyzed the imaging data and quantify the co-localization with CD63 (Fig. 4B-E).
7. Along the same lines, the images in Fig4B with CD63 "show" some overlaps in yellow, but there are so many dots (background fluorescence?) with IQGAP1 that one wonders if the authors were to flip by 180 degrees yellow and green images (randomizing) would the number of yellow dots remain the same... No quantification/statistics of any kind is a big issue here.
We have in fact had a control staining using IQGAP1 knockout cells and verified that the detected signals were indeed specific to IQGAP1 (Fig. VII in this document). We thank the reviewer noting the lack of quantification and statistics. We have re-analyzed the image and quantify accordingly (Fig. 4B-E).
8. TSG101 is an upstream ESCRT in ESCRT machinery which recognizes the cargo protein but doesn't directly regulate the membrane remodeling. Did authors check other ESCRT proteins?
The membrane remodeling process is primarily mediated by the ESCRT III complex, which consists of multiple essential and a few non-essential subunits. Knockdown of Vps24, a component of ESCRT-3 that catalyzes the last step of membrane remodeling during ILV formation, suppressed the release of polyubiquitinated pro-IL-1β and extracellular vesicles (Fig. EV3A-B), further demonstrating a critical role of the ESCRT system in this process.
9. In the abstract the authors mention that IQGAP1 bridges GSDMD to ESCRT machinery to promote IL-1β containing exosomes in response to NLRP3 inflammasome activation but haven't shown any data on NLRP3 in this study.
We agree with the reviewer wholeheartedly on this matter. Using MCC950, a small molecule that inhibits NLRP3 oligomerization, we found that inhibition of NLRP3 inflammasome activation abolished the production of polyubiquitinated pro-IL-1 and sEVs production in response to LPS and ATP stimulation (Fig. EV1B). Furthermore, we found that inhibition of caspase 8 but not caspases 1 attenuated the production of poly-ubiquitinated pro-IL-1β and sEVs (Fig. EV1C). Taken together, the data indicate the LPS and ATP stimulation activated NLRP3casapse 8 inflammasome in the YAMCs.

Is IL-1β biologically active?
Our data suggest that the IL-1β released through the exosomal pathway is active. Please see our response to point #2.

If increased in exosomes, how does IL-1β contact the receptors on cells to achieve its biological activity?
The question the reviewer raised touches upon an understudied aspect of exosome biology in general, namely how exosome delivers its cargo to the destination. We respectfully think this aspect is beyond the scope of the current study. We agree that it would be very important to elucidate this mechanism and we do have plans to address it in the future.
12. There are some known paradoxical relationship with GTP and GDP states known regarding IQGAPs and Rho GTPases. It seems that the authors may not be aware of those and believe that GTP state (typically associated with "active" small GTPases) is the ON state. This needs to be resolved. Also, IQGAPs are calmodcuin binding protins and Ca2+ may play a role... We thank the reviewer for pointing our inaccurate phrasing in our text when we referred to the literature. Indeed, IQGAP1 does not only interact with GTP-bound small GTPases. For instance, IQGAP1 preferentially binds to GDP-bound Rab27a (Kimura, Yamaoka et al., 2013). However, it is important to point out that the formation of GDP-Rab27a-IQGAP1 is also dependent on GTP-CDC42, highlighting the crucial role of GTP-CDC42 in regulating IQGAP1 effector function. This remains consistent with the well-established concept that GTP-CDC42 alters the conformation of IQGAP1, which has been verified by structural analysis of the complex (LeCour, Boyapati et al., 2016).
In response to the reviewer's suggestion, we explored the interaction between IQGAP1-GSDMD and IQGAP1calmodulin. We found that while GSDMD co-precipitated with IQGAP1, calmodulin was not detected in the same complex (Fig VIII in this document). Based on this, we deduce that calmodulin is not in the same complex with IQGAP1-GSDMD. This likely reflects the fact that both calmodulin and GSDMD binds to the IQ motifs in IQGAP1, forming mutually exclusive complexes. Therefore, we summarize that calmodulin is probably not directly involved in regulating the exosomal release of pro-IL-1β. On the other hand, using the chelator BAPTA-AM, we did find that calcium signal is required for the production of polyubiquitinated pro-IL-1β (Fig. EV3E). Given the established role of calcium signal in activating the ESCRT system, the data provides additional supportive evidence for our proposed mechanism. Figure VIII. YAMC cells were treated as indicated and whole cell lysates were subjected to co-immunoprecipitation with anti-GSDMD antibody followed by western blot analysis of indicated proteins.
Minor: 1. Please check the sizes of graphs in figure 3A We have resized the graphs as requested by the reviewer.
2. Figure 3E and F and figure 4F graphs are represented differently.
We thank the reviewer for noting the style issues and have rectified accordingly.
3. In figure 6A author haven't pointed out specific structure to look.
Arrows now point out the staining pattern in the revised manuscript.
4. On any of the western blot author haven't shown the marker to represent the specific size of the proteins.
We thank the reviewer for noting the minor negligence. Markers are added throughout the figures.

Referee #2
Liao et al. (the authors) identify IQGAP1 as a potential interaction partner of gasdermin-D (GSDMD). They show that interaction between IQGAP1 and the C-terminal region of GSDMD is required for secretion of exosomes from YAMC cells upon treatment with LPS+ATP. These exosomes(or the intracellular complexes preceding secretion) contain IQGAP1, GSDMD and among other components TSG101 and activated CDC42. These complexes assemble in CD63 positive endosomes. Ablation of TSG101 expression or inhibition of CDC42 reduced secretion of exosomes upon LPS&ATP treatment. Furthermore, stable assembly of the CDC42, IQGAP1, GSDMD complex requires presence of both IQGAP1 or GSDMD, suggesting a stabilizing function for either proteins. The authors finish their study with the demonstration that exosome secretion is reduced in colonic explants from IQGAP1 or GSDMD KO treated with DSS.
I think the data presented are very interesting and propose a non-canonical role for GSDMD in IL-1 secretion (besides pore formation). The paper is well written, and the experiments are very well controlled. I think most of the conclusion presented are substantiated by the data presented (except detailed below).
My major concern is regarding the upstream events leading to IL-1 secretion in exosomes. We know that stimulation of cells with ATP leads to (despite other events) opening of the pannexin-1 channel by P2X7 stimulation and ion fluxes across the plasma membrane, which are responsible for inflammasome activation. The authors show the presence of cleaved IL-1 beta in the supernatant but cleavage of GSDMD can't be observed (or at least not shown in the study). We don't know if YAMC do activate NLRP3 and if all the processes shown are dependent on inflammasome activation, although the authors claim this in the text. Calcium influx across pannexin-1 channels can be envisioned to lead to activation of CDC42. IQGAP1 is, after all, a calcium responsive protein. Some experiment I would suggest to address questions concerning this knowledge gap in the study: 1. Use MCC950 or 90 mM extracellular KCl to check if secretion of exosomes is dependent on NLRP3 activation 2. Use zVAD-fmk or qVD to check if secretion is dependent on caspase-1 activation In response to points #1 and #2: We agree with the reviewer wholeheartedly on this matter. Because we used classical NLRP3 inflammasome activators as stimuli (LPS plus ATP). We found that MCC950 profoundly blocked the production of poly-ubiquitinated pro-IL-1β and sEVs (Fig. EV1B).
Since we only detected active caspase 8 but not active caspase 1 from LPS-and ATP-treated YAMCs (Bulek et al., 2020), we employed more specific caspase inhibitors rather than a pan caspase inhibitor. Consistent with our prior report, we found that inhibition of caspase 8 but not caspases 1 attenuated the production of polyubiquitinated pro-IL-1β and sEVs (Fig. EV1C). Taken together, the data indicate the LPS and ATP stimulation activated NLRP3-casapse 8 inflammasome in the YAMCs.

Use extracellular EDTA or BAPTA-AM to check if exosome secretion or IQGAP1/GSDMD interaction is dependent on calcium influx
Following the reviewer's suggestion, we evaluated the impact of calcium chelation on the formation of IQGAP1-GSDMD complex and exosomal release of pro-IL-1β. We found that BAPTA-AM decreased the production of polyubiquitinated pro-IL-1β (Fig. EV3E).
Intracellular calcium mobilization has been long recognized as a trigger for ESCRT activation (Scheffer, Sreetama et al., 2014), which signals through calcium-binding protein-apoptosis-linked gene (ALG). Thus, the data from BAPTA-AM treatment serves to corroborate the involvement of ESCRT system in LPS and ATPinduced exosomal IL-1β release. Additionally, Ca 2+ surge is also linked to mitochondria dysfunction, which is required for NLRP3 inflammasome activation (Horng, 2014). Therefore, while the data are largely supportive of the involvement of Ca 2+ in the process, we hesitate to conclude that the impact of BAPTA-AM was due to a direct role of Ca 2+ on IQGAP1-GSDMD or IQGAP1-CDC42 complex formation. We limit our claim on this data in the manuscript, using it as supportive evidence only.
Minor suggestions: Figure 3: Can the authors show a PLA assay between IQGAP1 ∆IQ and GSDMD as an independent confirmation of abolished interaction?
We thank the reviewer for this suggestion. We performed the prescribed PLA and include the data in revision (Fig. 3C). The PLA confirmed the loss of interaction between IQGAP1 ∆IQ and GSDMD. Figure 4C/D: Quantification of PLA is missing. Is it possible to stain for pro-IL beta to see colocalization with CD63 in wildtype and IQGAP1 KO cells?
We thank the reviewer for noting the missing quantification. We performed the suggested staining and found that IQGAP1 deficiency alters pro-IL-1 trafficking to CD63+ puncta (Fig. 4E).

16th Aug 2022 1st Revision -Editorial Decision
Dear Kate, Thank you for submitting your manuscript to The EMBO Journal. Your study has now been seen by the original two referees and their comments are provided below.
As you can see both referees appreciate that the analysis has been extended and they appreciate the added changes. However, they also feel that some points have not been resolved well enough. I have looked at the comments and I agree with the referees that addressing these last points with experimental data would significantly strengthen the findings. Can we discuss how to best address the last points?
When you submit the revised version will you also please take care of the following editorial points.
-The figures and EV figures need to be uploaded as separate figure files.
-We are missing ORCID ID for Li and Zhao -Does the LC-MS/MS need to be deposited in a database? See also our guidelines to authors.
-Funding 1S10OD023436-01 is missing in the online submission system.
-We no longer have an author contribution section as we used the information that is provided in the online submission system. You can use the free text box in the system to add more information.
-COI needs to be updated as Disclosure Statement & Competing Interests -Please check the reference list if more than 10 authors use et al.
-The tables should be re-labelled in the MS text as Table EV1-3 -We don't encourage Data not shown (page 6). Please re-phrase -We now encourage the publication of source data for electrophoretic blots and quantitative data. See https://www.embopress.org/pb-assets/embo-site/Guide%20for%20SourceData%20Submission-1656066810500.pdf for how to upload and organize it -We include a synopsis of the paper (see http://emboj.embopress.org/). Please provide me with a general summary statement and 3-5 bullet points that capture the key findings of the paper.
-We also need a summary figure for the synopsis. The size should be 550 wide by  high (pixels). You can also use something from the figures if that is easier.
-Our publisher has also done their pre-publication check on your manuscript. When you log into the manuscript submission system you will see the file "Data Edited Manuscript file". Please look at the word file and the comments regarding the figure legends and respond to the issues.
Just contact me so that we can set up a time to discuss the needed revisions.

With best wishes
Karin Karin Dumstrei, PhD Senior Editor The EMBO Journal Use the link below to submit your revision: https://emboj.msubmit.net/cgi-bin/main.plex The authors have done extensive revision in many instances, and should be congratulated on the amount of work. However, on several key issues they chose to argue and maintain their position without further data. Of course, it is up to the editors to decide/adjudicate, but it is the duty of a responsible reviewer to point out such issues -as done below.
This reviewer's opinion remains that, while the manuscript has improved, there are important internal inconsistencies. Some of the key remaining ones are: 1) If exosomes are the way IL1 gets secreted, as the authors propose, then how come ESCRTs are not important -ESCRTs make instrumental vesicles that generate exosomes. 2) IQGAP1 is not known to function without calmodulin, and yet the authors state that GSDMD binds and displaces calmodulin. Does that mean that GSDM inactivate IQGAP?
3) The issue of "binding" remains unresolved. Many proteins can be found in large complexes, but a claimed (direct) binding needs to be demonstrated and cannot be based on a verbal argument that IQGAP is a large protein (it is not exactly too big). 4) The authors claim that the IL1 they are measuring as released is biologically active, but when asked about how the IL1 if it is trapped inside then exosomes can be biologically active they state that this is beyond the scope of the study. This reviewer's opinion is quite the opposite, that this issue is plumb in the center of the proposed process and should be left right and center within the scope of the study. 5) Given the point 4 above, it is possible if not likely that biologically active IL1 reported in the revision is a leak through pores in membrane (possibly generated by GSDMD, a known poreforming complex) paralleling exosome secretion as an epiphenomenon, and that exosomes are not the source of biologically active IL1.
This reviewer feels that before this work can be published it is important to address these questions, which were posed in the first round of review. This reviewer is willing to re-review the study if the above points can be adequately and whenever possible experimentally addressed. Of course it is up to the editor to decide.
Referee #2: I thank the authors for addressing my requests raised in the first round of peer review. I think the data presented is very convincing, however I feel there are some controls missing to fully support the conclusions made by the authors. 1. Biological activity of IL-1: The new data presented in figure EV1F is not well controlled. Yes, the secreted IL-1 has biological activity but we can't judge how much compared to IL-1 secreted by pyroptosis. The way the experiments needs to be performed is that supernatant from stimulated YAMC and BMDMS needs to be collected and the amount of IL-1 needs to be determined by ELISA. If the exosomes of YAMC cells need to be broken up, a low concentration detergent needs to be included in the measurement. Then, equal amounts of supernatant need to be added to the reporter cell line to conclude if the IL-1 secreted via the mechanisms described is bioactive. 2. Calcium: The authors state that they cannot make a definitive conclusion on the role of calcium on NLRP3 mediated shedding of exosomes. If BAPTA-AM really affects NLRP3 activation, as suggested by some publications (referenced by the authors), this should be visible by ASC speck formation as a proxy for NLRP3 activation. I think the authors should check ASC specks in the presence of BAPTA in YAMC cells to determine if calcium is required for assembly and secretion of the GSDMD/IQGAP1/CDC42 complex on exosomes 3. The new data interrogating IL-1 localization is very compelling. One could explain most observations presented with the fact that IL-1 fails to localize to secretory exosomes and that this is the function of IQGAP1 and ESCRT in this context. Do the authors have evidence that exosomes secretion in the absence of IL-1, GSDMD/IQGAP1 complex formation and recruitment to endosomes are unchanged?
We would like to thank the reviewers for their constructive criticism, all the comments and suggestion regarding our manuscript. Please find below our responses in blue font below each point.

Referee #1:
The authors have done extensive revision in many instances, and should be congratulated on the amount of work. However, on several key issues they chose to argue and maintain their position without further data. Of course, it is up to the editors to decide/adjudicate, but it is the duty of a responsible reviewer to point out such issues -as done below. This reviewer's opinion remains that, while the manuscript has improved, there are important internal inconsistencies.
We thank the reviewer for affirming the volume and improvement of our work during revision. We also greatly appreciate the reviewer for the thorough consideration of the data and for raising the remaining concerns. After careful digestion of the reviewer's comment, we feel that some of the remaining issues possibly stem from miscommunication/misunderstanding (we apologize in advance if our data presentation or writing did not clearly convey our message/conclusion). Additionally, we do also hold different opinions with regard to a few conceptual issues from the reviewer. We hope to use this opportunity to advocate for our findings and conclusions.
Some of the key remaining ones are: 1) If exosomes are the way IL1 gets secreted, as the authors propose, then how come ESCRTs are not important -ESCRTs make instrumental vesicles that generate exosomes.
We are claiming that ESCRT is required in the process based on data from knocking down of 2 different ESCRT components (Tsg101 and Vps24). To clarify, we are in total agreement with the reviewer that ESCRTs are important for IL-1β release via exosome.
2) IQGAP1 is not known to function without calmodulin, and yet the authors state that GSDMD binds and displaces calmodulin. Does that mean that GSDM inactivate IQGAP?
We agree with the reviewer that calmodulin plays a crucial role as an IQGAP1 modulator. It is not our contention to propose a calmodulin-independent function of IQGAP1.
Since we did not detect calmodulin in the same complex with GSDMD, we feel that any connection with calmodulin would likely be an indirect or secondary process that deserves its own dedicated study rather than an add-on to the current manuscript.
Notably, calmodulin was shown to disrupt IQGAP1-CAC42 interaction. Calmodulin clearly changes IQGAP1 conformation and modulates its binding partners in a Ca 2+ responsive way. But there is no evidence to suggest that IQGAP1-calmodulin complex represents a functionally active complex. https://www.jbc.org/article/S0021-9258(18)37108-4/fulltext In other words, the absence of calmodulin in the GSDMD-IQGAP1 does not mean "inactivation of IQGAP1".
3) The issue of "binding" remains unresolved. Many proteins can be found in large complexes, but a claimed (direct) binding needs to be demonstrated and cannot be based on a verbal argument that IQGAP is a large protein (it is not exactly too big).
Co-immunoprecipitation is a very well-established methodology to detect ligand-induced formation of functional complexes. That is exactly what we have discovered and claimed for IQGAP1-GSDMD interaction. It often takes years and sometimes decades to sort out the direct and indirect interaction 25th Aug 2022 2nd Authors' Response to Reviewers and collaboration with structural biologists to solve the structure of the protein complex. Notably, identification of ligand-dependent functional complexes has led to the discovery of many signaling pathways, including TLR and IL-1 signaling cascades.
With close to 200kDa, the whole protein of IQGAP1 is a huge scaffold that provides binding platform that assembles huge protein complexes. Only fragments of the IQGAP1 structure has been resolved, the shape of the whole protein remains unknown. Importantly, the fragment that is responsible for interaction with GSDMD, the IQ motif, remains the unresolved section of IQGAP1, reflecting the inherent biochemical challenges in protein purification. Without high quality and purity recombinant protein (to which we do not have access), it would be imprudent to conduct biophysical interaction studies.
4) The authors claim that the IL1 they are measuring as released is biologically active, but when asked about how the IL1 if it is trapped inside then exosomes can be biologically active they state that this is beyond the scope of the study. This reviewer's opinion is quite the opposite, that this issue is plumb in the center of the proposed process and should be left right and center within the scope of the study. 5) Given the point 4 above, it is possible if not likely that biologically active IL1 reported in the revision is a leak through pores in membrane (possibly generated by GSDMD, a known poreforming complex) paralleling exosome secretion as an epiphenomenon, and that exosomes are not the source of biologically active IL1.
We strongly disagree with the reviewer's contention that our observation is merely an epiphenomenon and the process is associated with pore formation. In our opinion, we have supplied ample evidence that demonstrates a pore-forming independent IL-1 release process.
 We have shown that GSDMD cleavage by caspase 1 (Bulek et al, 2020) and its oligomerization (current manuscript) were both dispensable for GSDMD to mediate IQGAP1-dependent exosome production.  Both cleavage by caspase 1 and oligomerization are obligatory biochemical events for pyroptosis. The fact that exosomal IL-1release did not require either Caspase 1 cleavage nor GSDMD oligomerization, argues against the involvement of pores in the process.  Consistently, we did not detect any PI permeability in response to LPS+ATP in YAMCs, further showing that this process does not involve pore formation.  Lastly, we have also shown (current manuscript) that merely poking holes on plasma membrane is not sufficient to induce IL-1β polyubiquitination, a hallmark of exosome-mediated cargo release.
We also respectively disagree with the reviewer's notion that IL-1β is "trapped" in exosomes. We have provided biochemical, cellular and genetic evidence to indicate IL-1 as a cargo of exosome. Exosomes are emerging as a major conduit for cell-cell communication. Precisely how exosomes cargo is unloaded remains an active area of research. However, growing evidence indicates that the cargos do get unloaded and remain biologically active. Notably, we did provide new data to demonstrate that exosomal IL-1 is biologically active in our revision ( Fig. EV1A and EV1F). We do agree with the reviewer that it is important to understand the precise mechanism of cargo unloading allowing IL-1's recognition of the receptor on the target cells. We have several hypotheses about this, which will be a major undertaken, involving development of multiple new technologies in the lab (beyond the scope of this current study).
This reviewer feels that before this work can be published it is important to address these questions, which were posed in the first round of review. This reviewer is willing to re-review the study if the above points can be adequately and whenever possible experimentally addressed. Of course it is up to the editor to decide.
Referee #2: I thank the authors for addressing my requests raised in the first round of peer review. I think the data presented is very convincing, however I feel there are some controls missing to fully support the conclusions made by the authors.
We thank the reviewer for the positive comment and appreciate the highly constructive comments that helped to advance many key points of our study.
1. Biological activity of IL-1: The new data presented in figure EV1F is not well controlled. Yes, the secreted IL-1 has biological activity but we can't judge how much compared to IL-1 secreted by pyroptosis. The way the experiments needs to be performed is that supernatant from stimulated YAMC and BMDMS needs to be collected and the amount of IL-1 needs to be determined by ELISA.
If the exosomes of YAMC cells need to be broken up, a low concentration detergent needs to be included in the measurement. Then, equal amounts of supernatant need to be added to the reporter cell line to conclude if the IL-1 secreted via the mechanisms described is bioactive.
We appreciate the reviewer's suggestion and rationale. However, we have doubts about how such comparison would shed more light on the issue. BMDMs are professional pro-inflammatory cells capable of producing large amount of cytokines and with more robust inflammasome activation. Since ELISA does not discern mature vs pro-IL-1β, even if we normalize assay input based on total IL-1β, we may very well see that IL-1β produced by BMDMs inducing more NFB activation in the reporter assay. However, that evidence alone do not negate the fact that exosome preps from YAMC cells also induced IL-1β-dependent NFB activation.
2. Calcium: The authors state that they cannot make a definitive conclusion on the role of calcium on NLRP3 mediated shedding of exosomes. If BAPTA-AM really affects NLRP3 activation, as suggested by some publications (referenced by the authors), this should be visible by ASC speck formation as a proxy for NLRP3 activation. I think the authors should check ASC specks in the presence of BAPTA in YAMC cells to determine if calcium is required for assembly and secretion of the GSDMD/IQGAP1/CDC42 complex on exosomes While we agree with the reviewer that assays evaluating upstream events of inflammasome activation could help to verify whether the process was indeed inhibited by BAPTA-AM, we feel that such information may not provide more substantial insights into the process and advance the current study for a few reasons.  Firstly, our data show that BAPTA-AM did inhibit exosome production in response to LPS and ATP. It is likely BAPTA-AM treatment inhibited inflammasome activation (whether it's at ASC spec formation or more downstream remains to be investigated). However, such result would not contradict a possible role for Ca2+ signaling in regulating IQGAP1-CDC42 activation. Ca2+ signaling likely activates both pathways in a coordinated fashion to enable inflammasome assembly and prepare the intracellular vesicle trafficking system for its export.  Secondly, in an unlikely event that calcium signaling was dispensable for NLRP3 inflammasome in YAMC cells, that observation alone would warrant an independent study.
 Given the profound impact of calcium signaling, especially its well-documented role in inflammasome activation, our preference is not to make broad conclusions based on the evidence.
3. The new data interrogating IL-1 localization is very compelling. One could explain most observations presented with the fact that IL-1 fails to localize to secretory exosomes and that this is the function of IQGAP1 and ESCRT in this context. Do the authors have evidence that exosomes secretion in the absence of IL-1, GSDMD/IQGAP1 complex formation and recruitment to endosomes are unchanged?
In this study we are focusing on inflammasome-induced exosome production. We did not examine in great detail whether baseline exosome production is affected by GSDMD-IQGAP1 deficiency. However, we did not see any difference at baseline within 30 mins. But baseline exosome production in YAMC cells is small and our assays were not designed to capture the difference if there was any.
14th Sep 2022 2nd Revision -Editorial Decision Dear Kate, Thank you for submitting your revised manuscript to the EMBO Journal. Your revised manuscript has now been seen by referee #2. The referee appreciates your response but would also like to see some discussion regarding the points raised by referee #1 in the manuscript text -see below.
I think that is a good suggestion and would like to ask to do so in a final revision. I will accept the revised version once it comes back in. When you submit the revision will you also take care of the following pints: -We are missing ORCID ID for Li -Please correct the labelling of Tables EV1-3 in the legend as well -Please upload a "clean" version of the MS when you resubmit -The source data needs to be organised as one file per figure -see also https://www.embopress.org/pb-assets/embosite/Guide%20for%20SourceData%20Submission-1656066810500.pdf